Brucellosis antigens



Sept. 19, 1967 A. LEMBKE ETAL 3;342,684

BRUCELLOS I S ANT IGENS Filed July 2l, 1965 ATTORNEY United States Patent() 3,342,684 BRUCELLGSIS ANTIGENS Andreas Lembke and Karl-Ernst von Milczewski, both of Sielbeclr, near Eutin, Germany Filed July 21, 1965, Ser. No. 473,664 7 Claims. (Cl. 167-78) This application is 'a continuation-impart of our copending application Ser. No. 224,860, filed Sept. 19, 1962, now abandoned.

This invention relates to antigens capable of conferring immunity against brucellosis and to improved methods of preparing the same.

The causative organisms of brucellosis are Gram-negative bacilli. The recognized species which are known to be responsible for the disease are three in number. Brucella meltensis was isolated by Bruce in 1886 and shown to be the causative agent in Malta fever, contracted by drinking goats milk contaminated with these bacilli. Brucella abortus, characterized by Bang in 1897 is known to ybe responsible for contagious abortion in cattle. Brucella suis, identified by Traum in 1914, was isolated from aborting swine.

Control of brucellosis is a most complicated and diiicult problem. The disease spreads rapidly among cattle and infects humans secondarily by direct contact through the skin or through mucous membranes of the intestinal tract. For detection of the disease and destruction of infected animals considerable reliance is placed upon agglutination tests. Brieilly, Iagglutination is determined by mixing a series of dilutions of the test serum (obtained from an unvaccinated animal) with a standard suspension of killed Brucella organisms. The mixtures are incubated at 37 C. or 56 C. for several hours. A titre of 1:100 is strongly suggestive of Brucella infection.

Since there is no known method of chemical prophylaxis against brucellosis, vaccination of animals remains the most attractive method of control. Efforts in this direction Vhave continued unabated during the past thirty years, the prime target of research scientists being the preparation of a relatively non-toxic agglugen-free antigen capable of conferring immunity against brucellosis. Unfortunately, almost all brucellosis vaccines known to date are characterized by one feature of disadvantage or another, each being sufficiently detrimental to remove that particular product from the category of an optimally desirable immunizing agent.

The most effective brucellosis vaccine known today, from the standpoint of low virulence and high antigenicity, is Buck-19, identified by the United States Department of Agriculture as Strain 19. It is ecacious as a preventative of abortions and premature births. However, it has not succeeded in the removal of bovine brucellosis as a serious cattle malady. So-called persisting titres (agglutination titres) caused :by this procedure of immunization result in considerable diagnostic and epidemiological uncertainties, causing not a little diiiiculty in the control of infected livestock.4 Furthermore, newly vaccinated animals temporarily become bacteria carriers and are not distinguishable during this period from infected animals.

Ideally, in order to obtain an immunizing 'agent susceptible to rigid control standards and having optimal antigenicity yet lowest virulence and toxicity, the objective is to isolate from the Brucella organism a substantially pure, readily identifiable component. Moreover, the isolation of this component should be accomplished by methods which are relatively simple and may be easily duplicated by others.

Westphal et al. [2. Naturf. 7b, 148-155 (1952)] describe methods of extracting antigens from Gram-negative bacteria using phenol. One method is the so-called lCC warm phenol extraction process, wherein the organism is extracted between a phenol-saturated water phase and a water-saturated phenol phase, using high concentrations of phenol, and conducting the extraction at elevated temperatures. This method has not been entirely successful, it :being shown that species-speciiic antigens could not be obtained by this techniques. The phenol-saturated aqueous phase yields a lipopolysaccharide fraction including substantial quantities of nucleic acids (up to 50% in some cases), which fraction, moreover, is not characterized by strong antigenic power. The protein obtainable from the phenol phase exhibits some vagglutinogenic activity but this is primarily attributable to residual amounts of lipidlike material combined with sugar residues.

Another method is the so-called cold phenol extraction process, wherein the organism is extracted with aqueous phenol under reduced temperature conditions, The thus-obtained antigens, which are extracted from the phenol-saturated aqueous phase, are of high molecular weight, have relatively high sedimentation constants (about 175-200 S), cause the .formation of specific agglutinins, and .are highly pyrogenic, i.e.,I fever-producing. For example, the pyrogenic dose observed in mice for such antigens is about 0.002 microgram/kg. They are also considerably toxic, eg., antigen extracted according to this method from Proteus WA 68, injected intraperitoneally into mice, had an LD50 of 5--10l mg./kg.

In general, the cold phenol extraction process cornprises extracting bacterial organisms with a phenol-water mixture under reduced temperature conditions, and precipitating the antigenic fraction from the phenol-saturated aqueous phase with alcohol or acetone/alcohol. Separation of the upper aqueous layer from the lower phenolic layer after extraction is readily accomplished by centrifugation. lt is often advantageous to mix the phenolic layer with an additional portion of water, again separate the two layers, and pool the second aqueous fraction with the lirst. For further purification, dissolved phenol and lower molecular weight components may =be separated from the aqueous fractions by techniques such as dialysis against water, ultracentrifugation, adsorption (e.g., Sephadex), etc. Prior to precipitation of the antigen by addition of alcohol, the volume of the Iaqueous fractions is reduced to about one-third or one-half the original volume.

The concentration of phenol in the phenol-water mixture may be varied somewhat, it being necessary to keep in mind that one must ultimately obtain a Z-phase system, each phase being fully saturated with the other. For this purpose one may use equal volumes of water and phenol, the latter at a concentration from about 70% to about 95%. The preferred nal concentration of phenol is about The temperature employed during extraction is preferably as low as possible without attaining crystallization. Thus, one should operate at a temperature of about 1 C. to about 5 C., preferably about 2 C. As the lower alkanol for precipitation of the antigen, ethanol is preferred, although other alcohols of low molecular weight may be employed as, for example, methanol, propanol, isopropanol or butanol. Mixtures of lower alkanols and lower alkanones, e.g., acetone, are also suitable for this purpose. Also suitable as an aid in completing the extraction, is the addition of a small amount of sodium acetate.

It has now been found that, by means of a modified cold phenol extraction process, novel antigenic components may be obtained which are substantially pure substances possessing distinguishable physical and chemical characteristics, are capable of inducing immunity from brucellosis without producing conilicting titres following administration, i.e., they are substantially nonagglutinogenic, and, further, such components are substantially non-toxic and non-pyrogenic (fever-producting dose in mice: 1.0 microgram/kg. body weight). Prior to this time, antigens extracted from bacterial organisms with phenol have lacked one or more of these characteristics. The novel antigens herein may be obtained by (1) using organisms of the genus Brucella as the source of antigen and by (2) subjecting the phenol-saturated water phase to fractional precipitation with a chilled (15 C.) lower alkanol, without (3) the necessity of concentrating the volume of the aqueous fractions prior to such fractional precipitation. The alkanol is added, preferably about 1-4 parts by volume, and the resulting precipitate removed. Then a second portion of alkanol, preferably about 1-4 parts by volume, is added to the supernate, yielding the novel, antigenic fraction as a precipitate. The novel immunogen of this invention is designated hereinafter by the symbol A12. A further letter, e.g., S or is used to differentiate the immunogen as being extracted from a particular source of Brucella organism.

As the Brucella organism for the source of antigen, Brucella abortus, Brucella melitenss and Brucella suis may be advantageously employed, although Brucella abortus is preferred. Also includable Within the scope of the present invention, and intended to be so included, are strains, variants and mutants of the above-mentioned Brucella organisms which can be subjected to our process for the purpose of yielding antigenically active components. Of particular importance in this connection are mutants. These may be obtained as a result of applied phenomena such as heat, ultraviolet radiation or, perhaps, high energy radiation. Too, mutants are obtainable by growing Brucella organisms in an environment (nutrient medium) which, because of its constitution, suppresses the growth of normal Brucella organisms while at the same time inducing the formation and promoting the growth of mutants. One such mutant is identified as the MR form of Brucella abortus, strain Schaedtbeck, also designated as LM 6l, which yields the A12 antigen according to the present invention. this mutant is obtained by culturing Brucella abortus derived from the infected afterbirth of a cow on albumin-agar, and has been described previously (Doctoral Dissertation, Karl-Ernst von Milczewski, University of Kiel, Germany, 1960). The mutant is readily distinguishable `from known strains and forms of Brucella abortus by its morphological, cultural and other characteristics, which are:

(l) Resistance to fuchsine acid, thionine and pyronine; formation of urease, and color on staining procedures: similar to S-form of Brucella abortus Type I.

(2) Colonies of mucous-viscous consistency; diameter 2.10 mm. Edge: yellow, granular. Center: reddish yellow, strongly convex.

(3) Microscopically: smears from four days old tryptose agar culture show short rods, which tend to form chains (in contrast to cultures from S-form parent strain).

(4) Rapid and intensive formation of hydrogen sulfide.

(5) Trypafiavinagglutination: fine granular.

(6) Saline: unstable.

(7) Serologically: distinguishable from S-form of Brucella abortus by inagglutinability, i.e. not agglutinated by specific abortus Bang serum. Test animals subjected to preliminary treatment with LM 6l form no agglutinating antibodies to S-antigens but form such antibodies to the strain itself.

(8) In animal tests the strain is apathogenous.

(9) Serum rapid agglutination: negative.

For purpose of characterization, an inoculum of the mutant is introduced into tryptose agar (1.6% agar, 2% Difco-tryptose, pH 6.7) by means of the glass thread technique and the whole incubated at 37 C. for four days. The morphology of the colonies is determined by `means of a twelve-power magnification binocular microscope. Trypaavin-agglutination is done according to the d method of Braun and Bonnestel [Am. I. Vet. Res., 8, 386 l947)]. Rapid agglutination takes place with a 1:10 dilution of agglutinating Brucella antiserum. Borate buffered (0.1 M, pH 8.0) saline is used as diluent. The mixture of bacterial suspension and serum dilution is observed for agglutination on the microscope slide after 3 minutes. Agglutination with salt is carried out also (on the slide) using 0.9% saline. Using the staining technique of Hansen, the morphology of the cells is determined with the aid of the phase contract microscope under 900 magnification power.

Brucella abortus, strain Schaedtbeck, Form MR is obtained from .a culture of Brucella abortus, strain Schaedtbeck, Form R after thirty days incubation at 37 C. in forced tryptose as the only dissociant. The tryptose medium has the following composition: Tryptose 1.5%, glucose 0.5%, sodium chloride 0.5%, thiamine hydrochloride 0.5 mg.%, pH adjusted to 6.7 with phosphoric acid. The MR form mutant differs from the parent R form in that the latter shows the following morphological and culture characteristics:

(l) Colonies, after 4 days in tryptose agar: small; 1.70 mm. in diameter; edge, yellow and granular; center, reddish-yellow; consistency, granular-sticking.

(2) Trypaflavinagglutination: granular.

(3) Sodium chloride: unstable.

(4) Rapid serum agglutination: negative.

(5) Hansen stain: oval cells, tend to form long chains.

The mutant differs also from Brucella abortus, Strain 19, S-form in lthat the latter has the following morphological and culture characteristics, when propagated in the medium used for the R form (above) which elucidates the MR form mutant.

(l) Colonies after 4 days in tryptose agar: small; 1.4-1.6 mm. in diameter; edge, greenish-blue; center, faintly reddish-yellow; consistency, buttery deliquescent.

(2) Sodium chloride: stable.

(3) Trypaavinagglutination: negative.

(4) Rapid serum agglutination: rapid positive.

(5) Hansen stain: cells morphologically uniform, round, individual or in short chains.

It will be understood therefore, by way of -recapitulation, that the present invention includes within its intended scope, methods for producing antigens from various species of Brucella, as well as their known strains, variants, and mutants. It embraces also the useful antigens obtained by these methods, with particular attention focused on hitherto unoibtained antigenic components which have especially desirable utility as immunizing agents against brucellosis. Such -antigens tfall within the scope of the invention and will be mo-re yfully described hereinafter.

There is no valid Ibasis for correlation between the titre of agglutinins and the degree of acquired immunity in immunization against brucellosis. In our hands, agglutinogenic characteristics of brucellosis vaccines are not identical with immunizing characteristics. Therefore, we believe that animals may be immunized against brucellosis without simultaneous formation of agglutinins. In fact, agglutinin formation may be regarded as an avoidable and undesired side reaction. This is so because (1) lagglutinogenic and immunizing antigens are distinct from each other, or (2) both the agglutinogenic and immunizing antigens Iare combined on one and the same complexantigen-molecule but represent different determinative groupings. This problem is dealt with by means -of preparation of purified antigens directed toward separation of agglutinogens and immunogens. Thus, the object is to separate a purified immunizing antigen from Brucella abortus which is substantially non-agglutinogenic and non-toxic and which can be used as a prophylactic against the disease.

XTRACTIN AND ISOLATION OF ANTIGEN A1 A virulent strain of Brucella abortus, strain Schaedtbeck, S-form, is used for preparation of antigens, The bacteria are cultivated for a period of 48 -hours on a liver-extract agar prepared in the following manner: One kilogram of liver is skinned and cut to pieces. One liter of water is added and the mixture heated for 2 hours in a steam pot. The supernate is decanted and sterilized by being heated in a steam pot on each of three successive days. Forty-tive g. of Merck-Standard-I-agar are added to each 150 ml. of the broth thus obtained. Enough water is added to yield 1 liter of final product.

The cells grown on the liver-agar described above are Washed off with a phenol-saline solution (0.5% phenol, `0.85% sodium chloride) and sedimented by centrifugation. The cells are Washed twice with physiological saline, resuspended and filtered through a G2-filter (GZ-Fritte) in order to remove coarse particles from the bacterial cell suspension. The suspension is poured into acetone, the cells are sedimented by centrifugation and dried under reduced pressure after being washed several times with acetone.

Two -to three grams of acetone-dried bacteria are suspended in 80 ml. distilled Water and chilled to a temperature of -{-2 C. To this suspension are added 130 ml. of a 75% mixture of phenol/water (100 g. phenol +32 ml. water) previously chilled to +3 C. The mixture is thoroughly shaken and kept in the cold for a period Aof 20 to 30 minutes accompanied by occasional shaking. The phenol/Water suspension is disforced now by centrifugation. The supernate Water phase is decanted. Approximately 60 ml. of water are added to the remaining phenolic layer and sediment which are centrifuged after being thoroughly shaken. Both water layers are pooled. This is fraction A.

The water phase of the phenol extraction (fraction A) is dialyzed for la period of three day-s against tap -water and for one day against distilled Water. The volume of A is reduced to 30 to 40 ml. by vacuum distillation at approximately 30 C. The solution thus obtained shows weak opalescence. The solution is subjected to high-speed centrifugation. Enough chilled ethanol is added to the supernate to yield a nal concentration of 70% ethyl alcohol. A substance precipitates which hereinafter is identified as fraction A1 (Antigen A1). This fraction is sedimented by centrifugation, washed with ethyl alcohol, acetone and ether, and dried under reduced pressure to -give a yield in an amount equivalent to about 2% yield of dried bacterial matter.

OHARACTERIZATION OF ANTIGEN A1 Antigen A1 is a greyishabrown powder with relatively good solubility in Water. It responds to reactions listed below as indicated:

Molisch 'reaction for carbohydrates -l- Xanthroprotein reaction for amino acids containing aromatic structures Biuret reaction for proteins and polypeptides -i- By ultracentrifugation Antigen A1 shows even distribution with an accurate gradient. Spectral analysis in the range of ultraviolet light shows lack of absorption bands at 260 ma which are characteristic for nucleic acids. Thus the substance is free from nucleic acids and may be regarded as being relatively pure glycolipoprotein. Hydrolysis of measured amounts with N-HCl at 100 C. for a period of one hour, followed by removal of the protein `by means of meta-phosphoric acid, affords a means of estimating the sugar content of the samples in the supernate by the anthrone-sulfuric acid method. Estimation based upon a standard curve measured with pure glucose gives a polysaccharide content of 21.5- 24.8% glucose units. Fifty mg. of Antigen A1 are kept for four hours in 25 ml. of N-sulfuric acid at 100 C.

The hydrolyzed material is neutralized with barium hydroxide to precipitate the sulfuric acid and the phosphorylated sugar. The neutralized hydrolysate is subjected to centrifugation, the volume of the supernate reduced to about 2% of that of the starting material and used for chromatographical separation. The following sugar residues arev identified: galactose, glucose, mannose, xylose, amino sugars (demonstrated with Elson- Morgan-reagent) The presence of lipid components in Antigen A1,is determined by staining with Sudan black. The test is conducted as follows: The antigen is suspended in 0.5% di-sodium phosphate solution in an amount to yield 1% solution of the antigen. Aliquots of the suspension, corresponding to 20, 50 and lOOfy of the test compound, are applied to filter strips. A series of spots are obtained. The filter strips are soaked for three hours in an ethanol solution of Sudan black (a hot solution containing 60% ethanol, saturated with Sudan black B and ltered). After staining, the filter strips are washed twice for fifteen minutes each in 50% ethanol. The occurrence of grey black or brown-black colorizations of the spots indicates the presence of lipids. According to this test, Antigen A1 gives a -l--f--ireaction.

IMMUNOGENIC PROPERTIES OF' ANTIGEN A1 Antigen A1 is tested in comparison with acetone-dried Brucellae for its ability to reinforce an existing immunity against Brucella abortus. To produce the primary immunity CFW-mice are injected once only with an effective depot vaccine (concentration: 2.5 109 killed Brucella abortus, MR-form, adsorbed on 10 mg. A1203 suspended in 1 cc. distilled water). The second injection is then made with the solution or suspension of Antigen A1 or the acetone-dried Brucellae in physiological saline sol-ution. For both antigens to be tested and the comparative injection with acetone-dried Brucellae a test series of 60 animals is used; for each series test groups consisting of 15 animals each are simultaneously treated according to the following scheme, the Greek letter gamma, represented by the symbol 'y, signifying herein microgram(s).

Test Group I: First vaccination with 0.05 cc. of depot vaccine per mouse intracutaneously: ten days later a second vaccination with 20'y of the antigen to be tested intraperitoneally in 0.2 cc. of fluid.

Test Group II: First vaccination as for Group I. Ten days later a second vaccination with 10'y of the antigen to be tested intraperitoneally in 0.1 cc. of iiuid.

Test Group III: First vaccination as for Group I. Ten days later vaccination with 5v of the antigen to be tested intraperitoneally in 0.05 cc. of fluid.

Test Group IV: First vaccination as for Group I. No further vaccination.

Twenty two days after the first vaccination viz. 12 days after the second vaccination the test animals in each series are experimentally infected. For the infection a sixty-hour old tryptose agar culture of Brucella abortus, S-form, Schaedtbeck strain, is used.

Five mice receive 2.5 microorganisms per mouse, a further 5 mice receive 5 1O5 microorganisms per mouse and finally 5 mice receive 1 106 microorganisms per mouse, intravenously in each case'. Four days after the experimentally induced infection all test animals are killed, their spleens extirpated and smeared on agar dishes to reisolate the microorganisms. The number of successfully infected animals and the number of reisolated microorganisms per spleen are determined and classified according to the dates of inoculation and test infection. As criterion the average logarithm of the number of reisolated microorganisms per spleen for each test infection dose and each vaccination dose is established. This number, the degree of infection is given in the corresponding table besides the number of fully protected animals (=no reisolation of Brucella abortus from the spleen). Sec data, Tables I through III.

volume. Instead, the resulting aqueous solution is treated with 2 parts by volume of chilled ethanol and 1% by TABLE I Test Group I II III IV Antigen dose 20v/mouse 10V/mouse 'y/mouse (l1/mouse Test infection dose (Br. per mouse) 2.5)(105 5 105 1)(10 2.5X105 5 105 1X1()e 2.5 105 5X1()5 1X1()0 2.5)(105 5 105 1X105 No. of animals tested 5 5 5 5 5 5 5 5 5 5 5 No. of animals protected 3 2 1 0 2 2 3 0 0 0 0 0 Percentage of animals protect 60 40 20 0 40 40 60 O 0 O 0 0 Degree of infection: Log of reisolation per Spleen 0. 99 1. 87 2. 13 1. (56 1. 68 2. 04 0.96 2. 36 2. 17 3. 20 3. 31 3. 28 No. of animals tested 15 15 15 No. of animals protected G=40% 4=2G.7% 3=20% 0=0% Total No. of protected animals after vaccination with acetonedried Br.=13 out of 45 or 28.9%.

TABLE II Test Group I II III IV Antigen dose v/mouse IC7/mouse ylmouse Oy/mouse Test infection dose (Br. per mouse) 2.5)(105 5)(105 lXlO 2.5)(105 5 105 1 1Of 2.5 1O5 5 105 1)(10 2.5)(105 5 10s 1)(10 No. of animals tested 5 5 5 5 5 5 5 5 5 5 5 No. of animals protected 5 2 3 4 4 3 3 0 2 1 0 0 Percentage of animals protected 100 40 60 80 80 60 60 0 40 20 O Degree of infection: Log of reisolation 0 per spleen 0 1. 72 0. 78 0.51 0. 47 1. 32 0.74 1. 58 0.83 1. 26 2. 79 2. 7 No. of animals tested- 15 15 15 15 No. of animals protected 10=66.7% 11=73.5% 5=33.3% 1=6.7%

Total No. of protected animals after vaccination with Antigen A1=26 out of 45 or 57.8%.

1 No. of animals protected/No. of animals tested.

2 Standard deviation.

This shows that Antigen A1 in doses of 20, 10 and 5 micrograms can reinforce an existing immunity. Vaccination with A1 confers complete protection on a number of test'animals against a test infection with 2.5 105, 5X105, and l 106 Brucellae. The total number of animals protected against all vaccination doses and test infection doses is 26 out of 45, i.e., almost 58%.

In contrast to this, the total number of animals protected against all vaccination doses and test infection doses by the use of acetone-dried Brucellae as immunogenic agent is 13 out of 45, i.e., about 29%.

EXTRACTION AND ISOLATION OF ANTIGEN A128 21 grams of acetone-dried bacteria (Brucella abortus, strain Schaedtbeck, Sforrn) are suspended in 320 cc. of distilled Water and cooled to 2 C. To this suspension is added a mixture of 400 g. of phenol and 130 cc. of water, cooled to 2 C. Further working up is carried out as described for Antigen A1 (above), except that, after dialysis, the pooled phenol-saturated aqueous layers are not subjected to evaporation in order to decrease the volume of a saturated ethanolic sodium acetate solution and the resulting precipitated sediment is centrifuged oi. A further 2 parts by volume of lchilled ethanol and 1% by volume of the sodium acetate solution is then added to the supernate and the precipitating antigen, A12S, is harvested by centrifugation. The antigen may then by dried, for example, in a vacuum exsiccator. The yield of fraction A128 thus obtained amounts to about 2.5% (based on the weight of dried bacterial starting material). The presence of sodium acetate is not critical in the extraction of A12 and may be eliminated.

EXTRACTION AND ISOLATION OF ANTIGEN A12 Dried cells of Brucella abortus, strain Schaedtbeck, MR-form, are used as the bacterial starting material. To prepare the dried cells, a virulent strain of the bacteria are cultivated as usual on liver-broth-Standard-I-agar, in-

cubation being for 6-7 days at 37 C. Then the bacterial cells are Washed off from the agar surface with 0.35% saline and filtered through a G-l-Fritte filter in order to remove coarse particles from the bacterial cell suspension. Four parts by volume of chilled ethanol (2-4 C.)

and 10 ml./liter of a saturated ethanolic sodium acetate solution are added to the suspension. The suspension is allowed to stand in the cold until precipitation is complete. The cells are sedimented by centrifugation and dried under reduced pressure.

21 grams of dried bacteria are suspended in 320 cc. of distilled Water and cooled to 2 C. To this suspension is added a mixture of 400 g. of phenol and 130 cc. of water, cooled to 2 C. The mixture is `shaken and maintained in the cold for about 30 mins. with occasional shaking. The phenol-saturated water layer is separated after centrifugation. The water-saturated phenolic layer is then mixed with an additional 300 cc. of Water. The mixture is then centrifuged and the aqueous layer separated and pooled with the previously collected Water layer. Centrifugations are preferably conducted under cold conditions. The pooled phenol-saturated water layers are subjected to dialysis against distilled water for days, and then mixed with 1 part by volume of chilled ethanol and 1% by volume of a saturated ethanolic sodium acetate solution. The mixture is allowed to stand at cooled temperatures (1-5 C.) and the resulting precipitate is removed. An additional 3 parts by volume of chilled ethanol and 1% by volume of the sodium acetate solution is added. On standing in the cold, a precipitate (A12U) forms which is collected, e.g., by slow speed centrifugation (3500 r.p.m.) and dried under reduced air pressure (yield: 67%

CHARACTERIZATION OF ANTIGEN A128 Antigen A128 is a glycolipoprotein substantially free of nucleic acids (about 1% content) which responds to the reactions given below as indicated in the following In order to determine the nucleic acid content, spec- Y trophotornetric measurements are taken of a 0.1% or a 0.01% solution of the antigen in filled quartz containers l cm. in depth (E2611-E290=E nucleic acid). The concentration of nucleic acid is determined by comparison with adenosin-triphosphate standard curves.

For the qualitative determination of the sugar residues, mg. of the antigen is dissolved in 2 ml. of 1 M HZSO.; and hydrolyzed in a sealed ampule for 4 hrs. at 100 C., with occasional shaking. The hydrolysate thus obtained is diluted with an equal volume of water and the pH adjusted to 7.0 (neutral) with 1 M barium hydroxide. After minutes, the sample is centrifuged. The supernate siphoned ofr and its volume reduced to about 0.9 ml. by vacuum evaporation. Then an equal volume of acetone is added. The hydrolysate thus obtained is ready for chromatographic study. Separation of the sugar components are achieved by descending paper chromatography. The following materials are suitable for this purpose:

Paper: Schleicher-Schull, 2043b Mgl Solvent:

I. n-Butanol-acetone-water (5 :5 :1) II. n-Butanol-pyridine-water (6:413) Developed with:

(a) Aniline-phthalate (for aldehyde sugars) (b) 4-dimethylamino-benzaldehyde (for amino sugars) (c) Naphthol-resorcinol-trichloroacetic acid (for ketonic sugars) The running times are generally 17-20 hrs. The solvent butanol-acetone-water serves lfor tests on the content of ketonic sugars as well as for a better separation of the pentoses. On the other hand, a better separation of the aldehyde sugars is achieved by the solvent butanolpyridine-water. The latter also yields better results in the separation of the desoxy-sugars.

Approximations of the quantitative sugar contents are obtained by comparison of certain known amounts of reference sugars which are simultaneously subjected to 10 chromatography. Obviously, only those sugar residues of the antigenic glycolipoprotein are determined by this procedure which are completely cleaved to monosaccharides. However, a conception of the quantitative relations is obtained.

During the chromatographic determination, an unidentiable component which is chromatographically fast running is also observed.

The following table records the data. obtained on elemental analyses of Antigen A128 (two extractions):

AUS, percent A128, percent 5. 40-5. 42 5. 14-5. 16 2 1l2. 25 2. 44-2. 48 l -1. 96 1. 78-1. 83 5. 87-5. 98 5. 97-6. 03 H2O. 2. 70-2. 78 Volatiles. 5. 74-5. 74

CHARACTERIZATION OF ANTIGEN A12 Antigen A12 is a glycolipoprotein substantially free of nucleic acids (about 1% content) which responds to the reactions given below as indicated in the following table:

Solubility in water 1 g./ 100 ml. Nucleic acid content 1 -1.1%. Lipid content (Sudan black method) |--|-I|-I. Sugar content (glucose units) 2 41-46%.

Galactose About 10%.

Glucose About 2%.

Mannose About 1.5%.

Xylose About 1%. Amino sugars (demonstrated with Elson- Morgan-reagent) j+. Molisch reaction for carbohydrates: Xanthoprotein reaction for amino acids containing aromatic structures: Biuret reaction for proteins and polypeptides:

0 g;L8A(7verage `of four extractions: 0.86%, 1.6%, 1.0% and 2 Minimum and maximum of l3-e14.%, 4.445%, t1-42%.

During the chromatographic determinations for sugars, an unidentiable component which is chromatographically fast running is also observed as in the case of Antigen A128. The optical rotation, sedimentation constant, and U.V. and I.R. spectrums for A12 are the same as those for A128.

Elemental analysis of Antigen A12 reveals the following data:

four :determinations 45-416 Percent C 34.19-84.24 H 5.38-5.45 N 3 96-4.02 P 1 04-1.05 Na 4.77-4.82 H2O 3.10-3.19 Volatiles 632-635 IMMUNOGENIC PROPERTIES OF ANTIGEN A128 Antigen A128 is tested on CFW-mice for its immunizing power. In contrast to the test with Antigen A1, the immediate immunizing action of A12S is tested without previous vaccination with depot Vaccine. For this purpose 40 test animals from each test -group are simultaneously treated according to the following scheme:

Test Group I: First vaccination with 20fy of A128 per mouse intracutaneously in 0.05 cc. of fluid. Ten days later second vaccination with the same dose intracutaneously.

Test Group II: First vaccination with 2v of A128 per mouse intracutaneously in 0.05 cc. of iiuid. Ten days later second vaccination with the same dose intracutaneously.

Test Group III: No vaccination.

Twenty-four days after the first vaccination, viz. 14 days after the second vaccination the test animals from each test group are experimentally infected. For this infection a forty-eight-hour old tryptose agar culture of Brucella abortus, S-form, Schaedtbeck strain, is used. Ten mice from each test group receive 5 X104 microorganisms per mouse, a further mice receive 1 105 microorganisms per mouse, 10 mice receive 2 105 microorganisms per mouse and iinally 10 mice receive 5 105 microorganisms per mouse, all intravenously.

Four days `after the test infection all test animals are killed, their spleens extirpated and tests conducted as described for Antigen A1. See Table 1V.

No. of Positive Spleens Immunization, Number After 28 Days Dose ('y/kg.) of mice AizU AizS In determining whether the rate of infection may be influenced by simultaneous application of the immunogen (A12 in this instance) and of virulent Brucella abortus-S (5 101 i.p.) to CFW-mice, the data below is observed. Appreciable protection is recorded for dosages of 001:50 gamma/kg. body weight, although advantageous results are observedv up to 50 gamma/kg.

TABLE IV Test Group I II III Antigen dose- 20 f/mouse 2 q//mouse 0 'y/mouse With test infection dose of 5 104 Br., fully protected animals 50% (5/10) 100% (l0/10)... 60% (6/10). With test infection dose of 1 105 Br., fully protected animals 90% (t9/10)... 100% (l0/10)... 40% (4/10). With test infection dose of 2 105 Br., fully protected animals 90% (il/10)-.. 90% (S3/10)..." 20% (2/10). With test infection dose of 5)(105 Br., fully protected animals 0% (0/10) 20% (2/10). 0% (0/10). Total No. of animals protected against all test infection doses. 57% (2B/40).. 77% (3l/40).-.. 30% (l2/40 Table IV clearly shows that Antigen A128 has an active immunizing eiect. 2'y were more eiective than 207 of CoMPARATrvE IMMUNOGENIC PROPERTIES oF Ams AND A12 The table immediately below summarizes the results of an experiment performed on genetically pure CFW- mice which had been pretreated once with a dose of the glyco-lipid-protein antigens A12S and A12. The animals are challenged by intraperitoneal injections of 5 X104 virulent brucellae (Brucella abortus-S) injections being made on the 21st day of the experiment. All animals are killed on the 28th day; the spleens are removed and homogenized under aseptical conditions. Then streaks are prepared from this -material on tryptose agarplates. Evaluation is done after 4 days of incubation `at 37 C. As may be gathered from the table, optimal activity is observed after pretreatment with about 0.1-1.0 gamma/kg., although advantageous results are obtained with 0.01-50.0 gamma/ kg.

No. of Positive Spleens After- Dose of ANU ('y/kg.)

7 days 14 days 21 days 28 days CO MPARATIVE AGGLUTINATION TITERS The agglutinogenic action of Antigen A128 is tested on rabbits, agglutination titers being determined after one dose of antigen. Rabbits of the same litter, being 10 weeks of age and having a weight of 2 kg., serve as the experimental animals. One rabbit is used for each dose of antigen tested. The antigens are dissolved in sterile physiological saline and then injected into the marginal ear vein of the experimental animals. Blood letting, preparation of serum, and determination of the agglutination titer are done in the usual manner. The results of the eX- periment are tabulated below. For the sake of comparison, this table also contains values determined with acetonedried Br. abortus S and with Antigen A1.

Agglutination Titer (days after Dose, injection of antigen) 7 days 10 days AHS Administration of 2107 of A128 per kg. of rabbit does not lead to the formation of agglutinins. A slight and fast disappearing agglutinin formation is observed when 25';l of A12S/kg. is administered. An injection of 50';l of A12S/kg. results in a titer of only 1:50 on the 7th day. On the other hand, 507 of A1/ kg. results in an agglutination titer of 1:1280 on the 6th day. Injection iof 40'y of acetone-dried Brucella per kg. results in a titer of 1:320. According to these experimental results, Antigen A128 exhibits a much less pronounced agglutinogenic action than either A1 or acetone-dried Brucella. Similar results are obtained for Antigen A12 as for A128. This indicates that the glyoo-lipid-protein A12 is extensively freed from agglutinogenic components.

In another experiment, rabbits are intravenously injected with to 80 micrograms of A128 per rabbit as shown in the table below and agglutination titers are compared with those obtained from acetone-dried Brucella abortus-S cells. The test animals are 4 months of age and belong to the strain Vienna Whites. Second intravenous injections are made 3 weeks after the first vaccination with 5 micrograms of the same antigen per animal. In order to determine agglutination titers, blood is drawn from the marginal ear vein of the animals at the following intervals: 14 days after the iirst vaccination, and 2 and 3 weeks after the second vaccination.

The data illustrate the lack of toxicity for antigens A128 and A12 as compared with antigen A1. The LD50s of the former are in excess iof 200 mg./kg.

In summary, it may be stated that Antigen A1 and Antigen A12 possess strong immunogenic properties, as demonstrated above in laboratory animals, when compared with that of acetone-dried Brucella. The antigens are substantially pure glycolipoproteins which may be used to confer immunity against brucellosis infection. However, although A1 displays an immunizing action, it is still somewhat endotoxic and agglu'tinogenic. On the other hand, as shown hereinabove, the immunizing action is still maintained in Antigen A12 although the agglutinogenic and toxic components are absent.

The novel antigens of this invention are used in the form of sterile solutions or suspensions, as the case may be, in conventional carriers commonly used as vehicles for administration of biological products in the form of injectables. These may be, for example, distilled water, physiological saline, Le., sodium chloride in water in a concentration of from about 0.85 to about 0.90%. Aqueous mixtures of water and benzyl alcohol may be used as partial carriers in combination with other salts commonly employed in the preparation of injectable solutions such as, for example, salts of alkali and alkali earth metals attached to an anion such as a phosphate chloride, sulfate Agglutination Titell A uniform dose of 5 micrograms of the respective antigen is given on 2d vaccination to each oi the test animals; i.v. application.

TOXICITY OF ANTIGENS A1, A128 and A12 The toxicity of antigens A1, A128 and A12 is illustrated by the data below. Each antigen is dissolved in sterile physiological saline and the concentration adjusted so that the injecting quantity of 0.5 ml. contains the dosage (mg/kg. body Weight) indicated. Injections are made intraperitoneally into CFW-mice being 6-8 weeks of age. 1 i

No. of Dead Animals at- Dose (ma/ke.)

No. of Animals 74 hrs.

72 hrs.

96 hrs.

AHS 200 150 Aiz Dienen meno: Por

OOO OOO OHRA? OOO OOO4 Ol-h ODO OOO @HNA OOO OCC Or-l etc., these being understandably non-toxic in nature and fully compatible with animal physiology. Obviously, the essential requirement in the preparation of injectables containing the novel antigens is to make the final preparation isotonic, although this would not be an extremely important factor when injections are used in large animals. In the case of small animals, however, this factor should be kept in mind. In this connection, it may `be desirable to combine the antigen with a small quantity of local anaesthetic which is compatible with the antigenic components, such as procaine hydrochloride. The concentration `of antigen in the final preparation is determined by known standardization techniques, said preparations preferably containing per dosage unit the desired therapeutically useful amount of antigen.

We have described herein techniques showing the separation of an immunogenic factor from Brucella organisms in substantially pure form, separated from the less desirable agglutinogenic and toxic components which are not factors in inducing immunity to the animal. Therefore, the degree of purity of the immunogen which is achieved when the processes of this invention are followed will reiiect in a large part in the antigen concentration of the iinal preparation. From this, obvious standardization techniques are the necessary and nally determinative factor in the preparation of the product. Examples of useful antigenic preparations are given hereinabove by way of illustration, but without intention to limit in any way the nature of the final product with respect to concentration of the antigen.

What is claimed is:

1. A substantially non-toxic and non-agglutinogenic immunogenic substance, identified as Antigen A12, which is useful in conferring immunity against brucellosis, in a physiologically acceptable injectable carrier, sald 1mmunogenic substance being a substantially pure glycolipoprotein characterized by the following properties: soluble an elemental analysis of about 30S-34.5% carbon, about 5.0-5.5% hydrogen, about 2.0-4.0% nitrogen, about 1.0- 2.0% phosphorus, about 4.5-6.0% sodium, about 2.0- 4.0% H2O and about 6.0-8.0% volatiles; shows only a weak maximum of absorption at 258 mit in the ultraviolet region of the spectrum, and in the infrared region exhibits no characteristic sharp bands; a sedimentation constant (1% solution in 1/ 15 M phosphate, pH 7.0 of s20=1.4- 1.5S; insignificant agglutination titres in rabbits at 5-80 micrograms/kg. body Weight; and an LD50 in rnice 200 mg./ kg. body weight.

2. In the method of extracting gram-negative bacterial organisms comprising the steps of treating said organisms with a phenol-water mixture containing 70-95 percent phenol at 1-5 C. for at least about 20-30 minutes, separating the phenolic phase from the aqueous phase, contacting said aqueous phase with a lower alkanol at temperatures of 1-5 C., and separating the resultant precipitate, the improvement which consists in the steps of so extracting Brucella organisms and, after separation of the precipitate, contacting the aqueous supernate with additional lower alkanol at temperatures of 1-5 C., thereby precipitating a substantially non-toxic and non-'agglutinogenic immunogenic substance useful in conferring immunity against brucellosis.

3. The method of claim 2 wherein the Brucella organism is Brucella abortus.

4. The method of claim 2 wherein the Brucella organism is a mutant of Brucella abortus.

5. In the method of extracting gram-negative bacterial organisms comprising the steps of treating said organisms with a phenol-water mixture containing 70-95 percent phenol at 1-5 C. for at least about 20-30 minutes, separating the phenolic phase from the aqueous phase, treating the phenolic phase with additional water and again separating the phenolic phase from the aqueous phase, combining the two aqueous phases and dialyzing the combined aqueous fraction against water for a few days, concentrating the dialyzed aqueous fraction to about 1/3-1/2 the original volume, contacting the concentrated aqueous fraction with ethanol at temperatures of 1-5 C., and separating the resultant precipitate, the improvement which consists in the steps of using Brucella abortus organisms as the bacterial organism, and, after dialysis, fractionally precipitating the combined aqueous fraction without concentrating its volume by contacting said aqueous fraction with 1-4 parts by volume of ethanol at a temperature of 1-5 C., removing the resultant precipitate, and contacting the aqueous supernate with an additional 1-4 parts by volume of ethanol at a temperature ofl 1-5 C., thereby precipitating a substantially non-toxic and nonagglutinogenic immunogenic substance useful in confer ring immunity against brucellosis.

6. The method of claim 5 wherein the bacterial organism is Brucella abortus, strain Schaedtbeck, MR-form.

7. The method of claim 5 wherein the bacterial organism is Brucella abortus, strain Shaedtbeck, MR-form.

References Cited UNITED STATES PATENTS 12/1959 Behrens et al. 167-78 OTHER REFERENCES Braun et al.: A Phenol-Extracted Bacterial DNA, Nature (4598) pp. 1356-7, Dec. 14, 1957.

Braun et al.: DNA-Associated Antigens of Brucella abortus; in Proc., 5th Internat. Mtg. Biol. Standardization (Jerusalem), pp. 367-82 (1959).

Farthing: The Role of Bordetella Pertussisas an Adjuvant to Antibody Production, Brit. I Exp. Path. 42, pp. 614-622, December 1961.

Kabat et al.: Experimental Immunochemistry, 2nd ed. (1961), published 1961 by Charles C. Thomas Co., Springfield, Ill., chapter 45, Extraction of Antigens from Micro-Organisms, pp. 736-744, chapter 60, Bovine- Type Antigens, pp. 830-837.

Olitzki et al.: Active Immunization of Mice Against Experimental Brucella Infections by DNA Conjugated Proteins, in Proc., 5th Internat. Mtg. Biol. Standardization (Jerusalem), pp. 367-82 (1959).

Olitzki: The Antigenic Relationship Between Phenol Extracted Bacterial, DNA and Other Soluble Antigens of Brucellae Studied With the Aid of the Agar Gel Precipitation Technique, Brit. J. Exp. Path. 41, pp. 623-32, December 1960.

Palczuk et al.: Studies on DNA-ase-Sensitive Antigens of Brucella abortus by Complement-Fixation, Proc. Soc.v

LEWIS GOTTS, Primary Examiner.

S. K. ROSE, Assistant Examiner.

UNITED STATES PATENT UFFICE CERTIFICATE OF CORRECTION Patent No. 3,342,684 September I9, 1967 Andreas Lembke et al.

It is hereby Certified that error appears in the above numbered patent requiring correction and that the said Letters Patent should read as corrected below.

Column 3, line 4l, for "this" read This line 44, for "Germany" read West Germany columns 7 and 8, TABLE I, Column III, in the heading, for y/mouse" read 5 `y/mouse Column 9, ine Z0, for l g. 100 ml." read l g./IOO ml. column 16, line Il, for "MR-form" read S-form Signed and sealed this 22nd day of October 1968.

(SEAL) Attest:

EDWARD J. BRENNER Commissioner of Patents Edward M. Fletcher, Jr. Attestingr Officer 

1. A SUBSTANTIALLY NON-TOXIC AND NON-AGGLUTINOGENIC IMMONOGENIC SUBSTANCE, IDENTIFIED AS ANTIGEN A12, WHICH IS USEFUL IN CONFERRING IMMUNITY AGAINST BRUCELLOSIS, IN A PHYSIOLOGICALLY ACCEPTABLE INJECTABLE CARRIER, SAID IMMONOGENIC SUBSTANCE BEING A SUBSTANTIALLY PURE GLYCOLIPOPROTEIN CHARACTERIZED BY THE FOLLOWING PROPERTIES: SOLUBLE IN WATER (> 1 G./100 ML.); SUBSTANTIALLY FREE OF NUCLEIC ACIDS; POSITIVE RESPONSES TO THE MOLISCH REACTION OF CARBOHYDRATES, XANTHOPROTEIN REACTION FOR AMINO ACIDS CONTAINING AROMATIC STRUCTURES, AND BIURET REACTION FOR PROTEINS AND POLYPEPTIDES; STRONGLY POSITIVE RESPONSE FOR LIPID CONTENT; CONTAINING ABOUT 41-46% "GLUCOSE UNITS" AND CHROMATOGRAPHICALLY SHOWING APPROXIMATE SUGAR CONTENTS OF ABOUT 10% GALACTOSE, ABOUT 2% GLUCOSE, ABOUT 1.5% MANNOSE, ABOUT 1% XYLOSE, AN UNIDENTIFIABLE FAST-RUNING COMPONENT, AND A POSITIVE RESPONSE FOR AMINO-SUGARS; OPTICAL ROTATION (1.14% IN DISTILLED WATER) OF ($)20(**D)= +12(+ OR -1.2 DEG) AN ELEMENTAL ANALYSIS OF ABOUT 30.5-34.5% CARBON, ABOUT 5.0-5.5% HYDROGEN, ABOUT 2.0-4.0% NITROGEN, ABOUT 1.02.0% PHOSPHORUS, ABOUT 4.5-6.0% SODIUM, ABOUT 2.04.0% H2O AND ABOUT 6.0-8.0% VOLATILES; SHOWS ONLY A WEAK MAXIMUM OF ABSORPTION AT 258 MU IN THE ULTRAVIOLET REGION OF THE SPECTRUM, AND IN THE INFRARED REGION EXHIBITS NO CHARACTERISTIC SHARP BANDS; A SEDIMENTATION CONSTANT (1% SOLUTION IN 1/15 M PHOSPHATE, PH 7.0 OF S20=1.41.5S; INSIGNIFICANT AGGLUTINATION TITRES IN RABBITS AT 5-80 MICROGRAMS/KG. BODY WEIGHT; AND AN LD50 IN MICE >200 MG./KG. BODY WEIGHT. 